This invention relates to proteins having retinol dehydrogenase activity, such as 9-cis, 11-cis or 13-cis retinol dehydrogenase activity, or trans retinol dehydrogenase activity and which form complexes with a specific portion of a membrane receptor for plasma retinol-binding protein (RBP) expressed, e.g., in retinal pigment epithelium (RPE), and, more specifically; proteins having cis retinol dehydrogenase activity, such as 9 or 11-cis retinol dehydrogenase activity, which form complexes with RBP-binding membrane proteins such as the 63 kDa RBP binding membrane protein found in RPE cells. The invention also involves isolation of the proteins, as well as nucleic acid molecules coding p32 or complementary to coding sequences therefor, in addition to various applications of these materials.
Retinoids (vitamin A-derivatives) have important physiological functions in a variety of biological processes. During embryonic growth and development, as well as during growth and differentiation of adult organisms, retinoids act as hormones and participate in the regulation of gene expression in a number of cell types. See Lied et al. Trends Genet., 17:427-433 (1992). It is believed that these effects are mediated through two classes of nuclear ligand-controlled transcription factors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs), Benbrook et al., Nature, 333:669-672 (1988); Brand et al., Nature, 332:850-853 (1988); Giguere et al., Nature, 330:624-629 (1987); Mangelsdorf et al., Nature, 345:224-229 (1990); Mangelsdorf, et al. Genes Dev. 6: 329-344 (1992); Petkovich et al. Nature 330:440-450 (1987); and Zelent et al., Nature 339:714-717 (1989).
Apart from their role as hormones in cellular growth and differentiation, retinoids are also involved in the visual process as the stereo isomer of retinaldehyde, 11-cis retinaldehyde, is the chromophore of the visual pigments. See, e.g. Bridges, The Retinoids,. Vol. 2, pp 125-176, Academic Press, Orlando, Fla., (1984).
Under normal physiological conditions most cells, both ocular and non-ocular, obtain all-trans retinol as their major source of retinoids. Despite the many different metabolic events taking place in different tissues, it is known that a common extracellular transport machinery for retinol has evolved. Specifically, in plasma, retinol is transported by plasma retinal binding protein (RBP). See Goodman et al., The Retinoids, Academic Press, Orlando Fla., Volume 2, pp. 41-88 (1984). The active derivatives of retinal, i.e., retinoic acid in non-ocular tissues and mostly 11-cis retinaldehyde for ocular tissues, are then generated by cellular conversion using specific mechanisms. To date, none of these mechanisms have been fully defined at the molecular level and several of the enzymes involved have only been identified by enzymatic activities. See Lion et al., Biochem. Biophys. Acta. 384:283-292 (1975); Zimmermann et al., Exp. Eye Res. 21:325-332 (1975); Zimmermann, Exp. Eye Res. 23:159-164 (1976) and Posch et al., Biochemistry 30:6224-6230 (1991).
Polarized retinal pigment epithelial cells (RPE) are unique with regard to retinoid uptake since all-trans retinol enters these cells via two different mechanisms. Retinol accumulated from RBP is taken up through the basolateral plasma membrane, while all-trans retinal, presumably taken up from the interstitial retinol-binding protein (IRBP) following bleaching of the visual pigments, may enter through the apical plasma membrane. See Bok et al., Exp. Eye Res. 22:395-402 (1976); Alder et al., Biochem. Biophys. Res. Commun. 108:1601-1608 (1982); Lai et al., Nature 298:848-849 (1982); and Inu et al., Vision Res. 22:1457-1468 (1982).
The transfer of retinol from RBP to cells is not fully understood. In a number of cell types, including RPE, specific membrane receptors for RBP have been identified, which is consistent with a receptor-mediated uptake mechanism for retinol. For example, isolated retinol binding protein receptors, nucleic acid molecule coding for these receptors and antibodies binding to the receptor have been taught, in references relating to the first of the two mechanisms. See Bavik et al., J. Biol. Chem. 266:14978-14985 (1991); Bavik, et al. J. Biol. Chem. 267:23035-23042 1992; Bavik et al., J. Biol. Chem. 267:20-540-20546 (1993); and copending U.S. Application Ser. No. 083,539 and International Publication WO 93/23538, all of which are incorporated by reference herein. See also Heller, J. Biol. Chem. 250:3613-3619 (1975); and Bok et al., Exp. Eye Res. 22:395-402 (1976).
Retinol uptake on the apical side of the RPE for the regeneration of 11-cis retinaldehyde is less well characterized. Regardless of the origin of all-trans retinol, however, the synthesis and apical secretion of 11-cis retinaldehyde seems to be the major pathway for accumulated retinol in the RPE. At present, it is not known whether similar mechanisms are used with regard to cellular retinol uptake through the basolateral and the apical plasma membranes. Available data do show that functional receptors for RBP are exclusively expressed on the basolateral plasma membrane of RPE-cells. Bok et al., Exp. Eye Res. 22:395-402 (1976).
It is also known that pigment RPEs express a 63 kDa protein (p63). This molecular weight, and all others, is by the refernce to SDS-PAGE, unless stated otherwise. It has also been shown by chemical cross-linking that this protein may be part of an oligomeric protein complex which functions as a membrane receptor for plasma retinol-binding protein (RBP) in RPEs, or a component of the retinoid uptake machinery in RPE cells. See Bavik et al, J. Biol. Chem. 266:14978-14875 (1991); Bavik et al., J. Biol, Chem. 267:23035-23042 (1992), and U.S. Application Ser. No. 083,539 and PCT application WO93/23538. The p63 protein has been isolated and the corresponding cDNA cloned. See Bavik et al., J. Biol. Chem. 267:20540-20546 (1993). Homologous molecules which bind to retinol binding proteins have been identified in other tissues, such as the liver. However, there is nothing in these references suggesting the existence of the protein which is a feature of this invention.
In accordance with this invention, proteins having retinol dehydrogenase activity which have a molecular weight of from about 30 to about 36 kD, as determined by SDS-PAGE, have now been discovered. These proteins form oligomeric protein complexes with the previous described components of the membrane receptor for RBP, such as the p63 molecule discussed supra. Also disclosed are nucleic acid molecules which code for these proteins. Sequence analysis shows that the proteins belong to the family of short chain alcohol dehydrogenases, and exhibit cis retinal dehydrogenase activity such as 9 or 11-cis retinol activity, the enzyme which catalyzes the stereospecific conversion of 9 and/or 11-cis-retinol into corresponding retinaldehydes in the presence of cofactor NAD+. In addition, other members of the short chain alcohol dehydrogenase family are disclosed, which have all trans-retinol dehydrogenase activity.
As will be shown, the proteins have many important uses. For example, owing to its membrane bound retinol dehydrogenase activity, which catalyzes the conversion of cis-retinols to cis-retinaldehydes, a major metabolic step in retinoid metabolism in RPE-cells, retinoid accumulation and metabolism which may lead to retinitis pigmentosa, may be directly or indirectly tied to the presence of these proteins, and/or their activation or inhibition. As the proteins have also been found to be members of the short chain alcohol dehydrogenase super family, many known alcohol dehydrogenase inhibitors (and activators) are available to develop activity assays, and thus diagnostic materials for retinol uptaken and retinoid metabolism.
Also a part of this invention are nucleic acid molecules which encode mammalian forms of the proteins, such as the human, bovine, and murine forms. Also a part of the invention are probes, based upon the nucleotide sequences described herein.
These and other aspects of this invention are more fully discussed in the following Detailed Discussion with accompanying drawings.